Cloning and expression of a sorghum gene with homology to maize vp1. Its potential involvement in pre-harvest sprouting resistance.

Pre-harvest sprouting (PHS) in sorghum is related to the lack of a normal dormancy level during seed development and maturation. Based on previous evidence that seed dormancy in maize is controlled by the vp1 gene, we used a PCR-based approach to isolate two Sorghum bicolor genomic and cDNA clones from two genotypes exhibiting different PHS behaviour and sensitivity to abscisic acid (ABA). The two 699 amino acid predicted protein sequences differ in two residues at positions 341 (Gly or Cys within the repression domain) and 448 (Pro or Ser) and show over 80, 70 and 60% homology to maize, rice and oat VP1 proteins respectively. Expression analysis of the sorghum vp1 gene in the two lines shows a slightly higher level of vp1 mRNA in the embryos susceptible to PHS than in those resistant to PHS during embryogenesis. However, timing of expression was different between these genotypes during this developmental process. Whereas for the former the main peak of expression was observed at 20 days after pollination (DAP), the peak in the latter was found at later developmental stages when seed maturation was almost complete. Under favourable germination conditions and in the presence of fluridone (an inhibitor of ABA biosynthesis), sorghum vp1 mRNA showed to be consistently correlated with sensitivity to ABA but not with ABA content and dormancy.

The TATA-less promoter of VP1, a plant gene controlling seed germination.

Vp1 is a seed-specific gene involved in the control of dormancy and germination. We here present the complete sequence of the sorghum vp1 promoter/enhancer region highlighting its main features, especially the lack of canonical TATA and CAAT boxes and the presence of elements responsive to abscisic acid and light. The region closest to the start of transcription is highly homologous to the partial proximal sequence reported for the maize vp1 promoter. This region is interrupted by a 57-nt stretch containing 14 CT microsatellite repeats. We observed a poor overall homology to the promoter from abi3 gene, the Arabidopsis counterpart bearing a similar coding sequence. However, there exists a high degree of homology (89%) between a TATA-rich 103-bp stretch of the sorghum vp1 promoter located about 700 nt upstream of the startpoint and miniature inverted transposable elements (MITEs) interspersed within the sorghum seed-specific kafirin cluster. This sorghum MITE-like element displays considerable homology (68%) to the TATA-less promoter from the sorghum NADP-malate dehydrogenase gene and lesser similarity to the Tourist, Pilgrim and Batuta MITEs previously identified within the promoter from the maize Abp1 (auxin-binding protein) gene.

Analysis of an abscisic acid (ABA)-responsive gene promoter belonging to the Asr gene family from tomato in homologous and heterologous systems.

Asr is a family of genes that maps to chromosome 4 of tomato. Asr2, a recently reported member of this family, is believed to be regulated by abscisic acid (ABA), stress and ripening. A genomic Asr2 clone has been fully sequenced, and candidate upstream regulatory elements have been identified. To prove that the promoter region is functional in vivo, we fused it upstream of the beta-glucuronidase (GUS) reporter gene. The resulting chimeric gene fusion was used for transient expression assays in papaya embryogenic calli and leaves. In addition, the same construct was used to produce transgenic tomato, papaya, tobacco, and potato plants. Asr2 upstream sequences showed promoter function in all of these systems. Under the experimental conditions tested, ABA stimulated GUS expression in papaya and tobacco, but not in tomato and potato systems.

Asr genes belong to a gene family comprising at least three closely linked loci on chromosome 4 in tomato.

Asr1, Asr2 and Asr3 are three homologous clones isolated from tomato whose expression is believed to be regulated by abscisic acid (ABA); the corresponding genes thus participate in physiological and developmental processes such as responses of leaf and root to water stress, and fruit ripening. In this report, results obtained with Near Isogenic Lines reveal that Asr1, Asr2 and Asr3 represent three different loci. In addition, we map these genes on the restriction fragment length polymorphism (RFLP) map of the tomato genome by using an F2 population derived from an interspecific hybrid cross L. esculentum x L. penelli. RFLP data allow us to map these genes on chromosome 4, suggesting that they belong to a gene family. The elucidation of the genomic organization of the Asr gene family may help in understanding the role of its members in the response to osmotic stress, as well as in fruit ripening, at the molecular level.

Sequence of Asr2, a member of a gene family from Lycopersicon esculentum encoding chromosomal proteins: homology to an intron of the polygalacturonase gene.

Asr is a family of genes regulated by abscisic acid, stress and ripening in tomato. Asr2, a recently reported member of this family, has been further characterized through sequencing of a genomic clone. We report the sequencing of 2029 bp of Asr2, spanning its AT-rich (62%) upstream region with probable regulatory functions. This region displays several candidate TATA and CAAT boxes that might be involved in transcription initiation, as well as a motif similar to one previously reported to be responsible for induction by ABA. Apart from that, we found a 108-bp stretch from the 3' non-coding region which displays a high homology (92%) to a region within intron 6 of the polygalacturonase gene, which, like Asr2, is expressed in ripening tomato fruit. This striking similarity suggests the presence of either a conserved regulatory motif or an abundant mobile element.

Identification of a major secretory glycoprotein from rat epididymis: interaction with spermatozoa.

A polypeptide with molecular mass of 17 kDa has been partially purified and identified as a major secretory glycoprotein in the rat epididymis. It is phosphorylated and contains high mannose-type oligosaccharides with 5 and 6 mannose units predominantly. These sugar residues are sufficiently exposed in the molecule to be released by endo-beta-N-acetylglucosaminidase H without prior denaturation or protease digestion. Specific binding of the glycoprotein to testicular spermatozoa was demonstrated with Ka 0.2 x 10(9) M-1 and 17,200 sites per cell, while no binding to epididymal spermatozoa was detectable. Direct labeling of surface proteins on cauda epididymis spermatozoa revealed the presence of a major band of 16.2 kDa, which may be equivalent to GP17. The interaction of the epididymal secretory protein with sperm suggests a possible role in the maturation process.

Androgen dependence of protein N-glycosylation in rat epididymis.

The rat epididymis is known to produce and secrete glycoproteins which interact with spermatozoa during the maturation process. The synthesis of the protein core of these compounds is dependent on androgenic stimulation. As a consequence, we studied the possible androgenic control of the N-glycosylation process dependent on the dolichol (Dol) pathway. Glucosyl and mannosyl transferase activities in rat epididymal microsomes decreased by approximately 76% after only 2 days of castration with respect to intact controls. Depleted mannosyl transferase activity could be restored to control values by administration of 100 micrograms/day testosterone propionate (TP) for 4 days. The effect of 20 micrograms/day TP was blocked by the simultaneous administration of 500 micrograms/day of the antiandrogen cyproterone acetate. The addition of excess dolichyl phosphate (12 times the Michaelis-Menten constant (Km) value) to the incubation mixture did not eliminate the difference in mannosyltransferase activity between epididymal microsomes from castrated rats and these from control or testosterone-treated animals. Moreover, the endogenous pool of dolichyl phosphate was found unchanged in the different hormonal situations. Finally, the incorporation of [14C]mannose into lipid-bound oligosaccharides and into glycoproteins was decreased by approximately 60% as a result of castration and reinduced to control values by treatment with TP (50 micrograms/day for 4 days). The results demonstrate the androgen dependence of the initial steps of N-glycosylation in the rat epididymis and suggest that the hormonal regulation is exerted at the level of Dol-nucleotide sugar transferases, rather than upon the size of the endogenous Dol phosphate pool.